The telomerase ribonucleoprotein (RNP) is required for maintaining telomeres, the specialized nucleoprotein structures that protect eukaryotic chromosome ends from aberrant processing and deleterious end-to-end fusion events. Telomerase catalyzes the processive extension of telomere DNA using a specialized catalytic mechanism that requires a strong functional interdependence of the telomerase RNA, telomerase reverse transcriptase (TERT), and several additional protein subunits. The primary objective of this proposal is to elucidate how conserved structural domains within telomerase RNA and protein subunits coordinate the processes of telomerase RNP assembly and catalysis. To address the substantial challenges associated with structural analysis of telomerase we will study the telomerase complex from the well-established model organism Tetrahymena thermophila, using a multifaceted experimental strategy that combines single molecule biophysical techniques paired with computational, biochemical, and high-resolution structural approaches. In aim 1, we will use chemical RNA probing and single molecule Forster resonance energy transfer (smFRET) to characterize the telomerase RNA solution structure and dynamics, respectively. Distance constraints that emerge from these experiments will be used to guide RNA structure prediction calculations in collaboration with Nikolai Ulyanov (UCSF). In aim 2, we will determine the three dimensional organization of conserved RNA and protein domains within the core telomerase RNP using targeted-hydroxyl radical probing, smFRET-based structure measurements, and x-ray crystallography. This work will be conducted in collaboration with Kathleen Collins (UCB) and Harry Noller (UCSC). In aim 3, we will exploit a novel single molecule telomerase structure-function assay to critically evaluate existing models for telomerase conformational dynamics during processive telomere DNA synthesis. In most cells, a progressive shortening of telomere length with each round of cell division provides a molecular signal for cell aging and regulates entry into permanent cell growth arrest. In contrast, cells possessing a high level of proliferative capacity (i.e. stem cells) maintain telomere length through the enzymatic action of telomerase. Understanding the molecular mechanism and regulation of telomerase is of direct medical significance because telomerase dysfunction contributes to human disease, including premature aging syndromes and the majority of cancers. Thus, telomerase research is motivated by the goal of developing novel approaches for diagnosing and treating telomerase-associated diseases. PUBLIC HEALTH RELEVANCE: Telomerase is an essential enzyme required for maintaining telomeres, the protective capping structures found at chromosome ends. Telomerase defects arise in the majority (~90%) of human cancers and several premature aging syndromes. Thus, efforts to better understand the mechanism and regulation of telomerase are motivated by the goal of developing novel approaches for diagnosing and treating telomerase-associated diseases. To this end, this proposal aims to illuminate the structural properties of telomerase that underlie its unique cellular activity.